This invention relates to the use of a polypeptide for determining the ability of an enzyme to modify the phosphorylation state of the polypeptide. Further aspects of the invention relate to a method for determining such an activity, and to a method for identifying substances which modify this ability of the enzyme.
Insulin is a peptide hormone which influences a large number of growth and metabolic pathways by binding to the insulin receptor and thus activating its intrinsic tyrosine kinase. This event leads to phosphorylation of a large number of proteins able to bind to the insulin receptor (IR), to specific tyrosine residues. The family of insulin receptor substrate (IRS) proteins also belongs to the proteins phosphorylated in this way.
Insulin receptor substrate 1 (IRS-1) is a cellular protein which can be phosphorylated by a large number of protein kinases (tyrosine-specific or serine/threonine-specific protein kinases) on tyrosine and/or serine residues and or threonine residues. It is assumed in this connection that there is specific phosphorylation of different tyrosine or serine/threonine residues depending on the enzyme. Apart from phosphorylation by tyrosine kinases such as, for example, apart from the insulin receptor (White 2002), the IGF-1 receptor (White 2002) or JAK 1/2 (Thirone et al. 1999), it is known that IRS-1 is also phosphorylated by serine/threonine kinases such as, for example, kinases from the PKC family (Schmitz-Peiffer 2002), inhibitor kappa B kinase complex (Gao et al. 2002), c-Jun NH(2)-terminal kinase (JNK, Aguirre et al. 2000) protein kinase A (Sun et al. 1991), mitogen-activated protein kinase (Mothe et al. 1996), protein kinase B (Paz et al. 1999), casein kinase (Tanasijevic et al. 1993), glycogen synthase kinase beta (Eldar-Finkelmann et al. 1997), AMP-activated kinase (Jakobsen et al. 2001) or phosphoinositol 3 kinase (Freund et al. 1995). IRS molecules are key molecules in the insulin signal transduction pathway and play a central role in maintenance of cellular functions such as growth, survival and metabolism. Phosphorylated IRS proteins serve in this connection as “docking” proteins with a large number of docking sites for the insulin receptor and with a complex network of intracellular signal molecules with so-called signal recognition complex (SRC) homology 2 domains (SH2 domains). Activation of these Sh2 domain proteins moreover activates certain signal cascades, which in turn leads to activation of various effectors which are located further downstream in the signal cascade, ultimately leading to transmission of the insulin signal to a branched series of other intracellular signal cascades (for review, see White 2002).
IRS belongs to a group of phosphoproteins which have a size of from 160 to 185 kDA and which serve as substrate of the insulin receptor. Four members of the IRS family (IRS-1, IRS-2, IRS-3 and IRS-4) are known. They differ in tissue distribution, subcellular localization, development-specific expression, nature of binding to the insulin receptor and nature of the SH2 proteins with which they interact. The four members of the IRS family have very similar structures in terms of their underlying protein structure: all have an amino (N)-terminal plextrin homology domain (PH domain) which binds to membrane phospholipids, a phosphotyrosine-binding domain (PTB domain) which is connected directly to the carboxy (C) terminus of the PH domain and is involved in the recognition of the Asp-Pro-Glu phosphotyrosine (NPEpY) sequence which is located in the juxtamembrane region of the insulin receptor beta subunit. They have moreover a somewhat less strongly conserved C-terminal part which has various potential tyrosine phosphorylation motifs to which specific SH2 domain-containing proteins can bind.
IRS-1 comprises 21 possible tyrosine phosphorylation sites, of which some are located in amino acid sequence motifs able to bind to the SH2 domain proteins. IRS-1 additionally comprises 30 potential serine/threonine phosphorylation sites in motifs which can be recognized by various kinases, such as, for example, kinases from the PKC family (Schmitz-Peiffer 2002), inhibitor kappa B kinase complex (Gao et al. 2002), c-Jun NH(2)-terminal kinase (JNK, Aguirre et al. 2000) protein kinase A (Sun et al. 1991), mitogen-activated protein kinase (Mothe et al. 1996), protein kinase B (Paz et al. 1999), casein kinase (Tanasijevic et al. 1993), glycogen synthase kinase beta (Eldar-Finkelmann et al. 1997), AMP-activated kinase (Jakobsen et al. 2001) or phosphoinositol 3 kinase (PI3 kinase, Freund et al. 1995). Inhibitory effects on the insulin receptor signal pathway can be explained at least in part by the recently discovered role of the serine/threonine phosphorylation of IRS-1, which is thought to be connected with an impairment of the interaction with the insulin receptor and/or a reduction in the in the tyrosine phosphorylation of IRS-1 and/or an impairment of the interaction with subsequent signal proteins able to bind to tyrosine-phosphorylated IRS-1 (for review, see White 2002). It has been possible to date to demonstrate for various kinases, for example kinases from the PKC family (Schmitz-Peiffer 2002), inhibitor kappa B kinase complex (Gao et al. 2002), c-Jun NH(2)-terminal kinase (JNK, Aguirre et al. 2000) protein kinase A (Sun et al. 1991), mitogen-activated protein kinase (Mothe et al. 1996), protein kinase B (Paz et al. 1999), casein kinase (Tanasijevic et al. 1993), glycogen synthase kinase beta (Eldar-Finkelmann et al. 1997) or phosphoinositol 3 kinase (Freund et al. 1995), that they phosphorylate IRS-1 directly in vitro. Moreover, in every case, an increased kinase activity in intact cells inhibited the activity of the insulin signal transduction pathway. In addition, the in vitro phosphorylation of RS-1 on serine/threonine residues was in some studies thought to be directly connected to the reduced tyrosine phosphorylation by the insulin receptor (Le Marchand-Brustel 1999)).
The sequences of IRS-1, 2, 3 and 4 are available to the public. The coding polynucleotide sequences and the relevant protein sequences of these genes can be accessed under the numbers NM—005544 (IRS-1 hs), XM—007095 (IRS-2 hs), NM—032074 (IRS-3 rat), NM—003604 (IRS-4 hs) from the NCBI nucleotide database.
NCBI is the National Center for Biotechnology Information (postal address: National Center for Biotechnology Information, National Library of Medicine, Building 38A, Bethesda, Md. 20894, USA; web address: www.ncbi.nhm.nih.gov). Cloning of the IRS-1 gene has been described inter alia in Araki et al. 1993 and Siemeister et. al, 1996; cloning of IRS-2 to 4 has been described by Araki et al 1994, Lavan et al. 1997a and Lavan et al. 1997b.
Various prior art methods are known for determining the ability and for measuring the activity of various kinases to phosphorylate IRS-1, the methods being based either on radioactive detection methods (e.g. transfer of radiolabeled phosphate to the substrate) or nonradioactive detection methods.
Thus, it is known to determine the phosphorylation of IRS-1 on full-length IRS-1 protein, fragments or peptides thereof which still have at least one phosphorylation site by a method in which radioactive phosphate residues are transferred to IRS-1 by incubation with radiolabeled ATP and the kinase to be tested as a function of the ability of the kinase to phosphorylate IRS-1. This is followed by chromatographic or electrophoretic fractionation of the IRS-1 and detection of the amount of transferred phosphate by flow scintillation or autoradiography (as described for example for the complete IRS-1 protein and glycogen synthase kinase 3 beta in Eldar-Finkelman et al. 1997, for a fragment of IRS-1 (amino acid 516-777) and insulin receptor, IGF-1 receptor or recombinant insulin receptor kinase in Siemeister et al. 1995 or an IRS-1 peptide (amino acid 601-616) with cell lysates which contain activated protein kinase from the PKC family in De Fea et al. 1997. In addition, it is from Siemeister et al. 1995 to determine the ability to phosphorylate IRS-1 fragments, for example a fragment of IRS-1 (amino acid 516-777) and insulin receptor, IGF-1 receptor or recombinant insulin receptor kinase by incubation with radiolabeled ATP, dropwise addition of the substrate to a positively charged membrane (nitrocellulose or similar material), washing and detection of the bound radiolabeled substrate by means of autoradiography or measurement of the radioactive emission.
Incubation of a biotinylated IRS-1 peptide (amino acid 601-616) with radiolabeled ATP, dropwise addition of the substrate to a streptavidin-coated membrane, washing and detection of the bound radiolabeled substrate by autoradiography or measurement of the radioactive emission is a further method for determining the ability of kinases to phosphorylate IRS-1 (see De Fea et al. 1997).
The disadvantage of the radioactive assay methods described above is obvious, because handling radioactivity entails considerable risks, is very costly and thus has low suitability in particular for high throughput methods (HTS methods).
The disadvantage of the methods described above which are based on the use of short peptides is that these peptides have unfavorable kinetic constants (Vmax, Km) and moreover the three-dimensional structure of peptides differs greatly from that of the physiological enzyme substrates. This is manifested on the one hand by a completely different folding, so that certain biological spaces which determine the specificity of the enzyme-substrate interaction are not present, which results either in a lack of recognition (and thus modification) or in a nonspecific recognition (and thus modification) and ultimately leads to incorrect results. Moreover, the shortness of peptides means that they have only one or a few phosphorylation sites, so that diverse peptides are necessary to investigate the phosphorylation modification of a particular substrate by different enzymes. This in turn also results in increased costs and an only conditional applicability for methods in the HTS format.
The object of the invention is therefore to provide a possible way of determining the activity of protein-phosphorylating and/or -dephosphorylating enzymes which does not have the abovementioned disadvantages.
This object is achieved by the use of a polypeptide (Def GGs to the peptide) to determine the ability of an enzyme, of a functional fragment or derivative thereof, to modify the phosphorylation status of a polypeptide, wherein the polypeptide is biotinylated.